Indole Test- Objective, Principle, Media, Procedure and Results

Indole Test- Objective, Principle, Media, Procedure and Results

Objective of Indole Test

To determine the ability of organism for the production of indole by the action of tryptophanase enzyme.

 Principle of Indole Test

Tryptophan, an essential amino acid, is oxidized by some bacteria by the action of trytophanase enzyme. Bacteria that produce the enzyme tryptophanase are able to degrade the amino acid tryptophan into pyruvic acid, ammonia, and indole. Indole is detected by combining with an application of reagent 4-dimethylaminobenzaldehyde, hydrochloric acid, and butanol, also referred to as Kovac’s reagent, which results in a formation of cherry red ring formation on the medium.

An alternative method uses Ehrlich’s reagent which has the same chemicals as the Kovac preparation, but it also contains absolute ethyl alcohol, making it flammable. Ehrlich’s reagent is more sensitive for detecting small amounts of indole.

Media used in Indole Test

SIM Medium

Composition per liter:

Pancreatic digest of casein = 20.0g

Peptic digest of animal tissue = 6.1g

Agar = 3.5g

Fe(NH4)2(SO4)2·6H2O = 0.2g

Na2S2O3·5H2O = 0.2g

pH 7.3 ± 0.2 at 25°C

Tryptone Broth

Composition per liter:

Pancreatic digest of casein = 10.0g

NaCl = 5.0g

pH 7.5 ± 0.2 at 25°C

Procedure of Indole Test

  1. Inoculate the organism into labeled tube by means of stab inoculation in SIM medium or inoculation into tryptone broth.
  2. Incubate the inoculated tubes at 37°C for 24-48 hours.
  3. Add 0.5 ml of kovac’s reagent and shake gently and allow to stand for some minutes.
  4. Observe for the formation of cherry red ring layer on the surface of the medium.

Result Interpretation of Indole Test

Result Interpretation of Indole Test

Positive: Pink- to cherry red colored ring after addition of Kovac’s reagent.

Negative: No color change after addition of the Kovac’s reagent.

Limitation of Indole Test

  • Ehrlich’s method may also be used to differentiate organisms under anaerobic conditions.

Quality Control of Indole Test

Kovac’s Method

Positive: Escherichia coli (ATCC25922)

Negative: Klebsiella pneumoniae (ATCC13883)

Ehrlich’s Method

Positive: Haemophilus influenzae (ATCC49766)

Negative: Haemophilus parainfluenzae (ATCC76901)

Ehrlich’s Method (Anaerobic)

Positive: Porphyromonas asaccharolytica (ATCC25260)

Negative: Bacteroides fragilis (ATCC25285)

Indole Test- Objective, Principle, Media, Procedure and Results

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